The mechanism of action of a variety of antimetabolite drug classes, most prominently those with anticancer activity, involves purine starvation; however, the mechanism of their biological cellular effects remains largely unknown. We have recently shown that purine biosynthesis inhibition results in a drastic reduction of DNA synthesis which is primarily caused by guanine, rather than adenine starvation. Consistent with this finding is the recent development of effective anticancer drugs that selectively inhibit IMP dehydrogenase and, thereby deplete only guanine. Moreover, DNA synthesis inhibition was paradoxically correlated with the depletion of GTP, rather than dGTP. Preliminary experiments aimed at resolving this paradox show that GTP depletion and DNA synthesis inhibition are associated with a selective reduction of the phosphorylation of histone H1. Modification of phosphorylation of this spacer histone is thought to affect DNA function and replication. Furthermore, labeling of histones in isolated cell nuclei with Gamma-32P-ATP and -GTP revealed that H1 is more efficiently phosphorylated with GTP than ATP. This proposal, therefore, tests the following hypotheses. i. GTP depletion causes a selective reduction of the phosphorylation of one or several sites on histone H1 or one of its variants. ii. The reduction of H1 phosphorylation by GTP depletion is linked to the inhibition of DNA synthesis. iii. A protein phosphokinase that utilized GTP may be involved in such regulation of DNA synthesis. Methods will include cell cultures, 32P labeling, enzyme isolation, peptide mapping, affinity labeling and cell mutant selection. One significant aspect of this proposed work would be the elucidation of a specific mechanism for a drug class that was formerly thought to act by a summation of multiple effects. This raises the possibility that cellular resistance to purine antimetabolites may occur through mutations affecting this specific mechanism. Moreover, this study could reveal a potential control mechanism of DNA synthesis that may normally serve to prevent cell division under unfavorable conditions.